Method for prediction of the progression risk of tumors

ABSTRACT

The present invention concerns a method for predicting the potential for aggressive growth and/or the risk to progress to high grade cancer for tumors in cell based detection procedures. In one aspect the invention concerns the detection of overexpression of cyclin-dependent kinase inhibitor gene products as a tool for predicting the progression risk and/or potential for aggressive growth of tumors. In a second aspect the invention concerns predicting the progression risk and/or potential for aggressive growth in tumors on the basis of the simultaneous co-detection of the presence of overexpression of cyclin-dependent kinase inhibitor gene products together with the expression of markers for active cell proliferation. Further the invention concerns preparations of probes for diagnosis namely for predicting the progression risk and/or the potential for aggressive growth of tumors.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. Ser. No. 14/249,259, filedApr. 9, 2014; which is a divisional of U.S. application Ser. No.13/062,185, filed Mar. 18, 2011, issued as U.S. Pat. No. 8,728,745 B2;which is a National Stage of International ApplicationPCT/EP2009/006415, filed Sep. 4, 2009, published Mar. 11, 2010, underPCT Article 21(2) in English; which claims the priority of EuropeanPatent Application No. 08105235.9, filed Sep. 4, 2008.

BACKGROUND Field of the Invention

The present invention concerns a method for predicting the potential foraggressive growth and/or the risk to progress to high grade cancer fortumors in cell based detection procedures. In one aspect the inventionconcerns the detection of overexpression of cyclin-dependent kinaseinhibitor gene products as a tool for predicting the progression riskand/or potential for aggressive growth of tumors. In a second aspect theinvention concerns predicting the progression risk and/or potential foraggressive growth in tumors on the basis of the simultaneousco-detection of the presence of overexpression of cyclin-dependentkinase inhibitor gene products together with the expression of markersfor active cell proliferation. Further the invention concernspreparations of probes for diagnosis namely for predicting theprogression risk and/or the potential for aggressive growth of tumors.

BRIEF DISCUSSION OF RELATED ART

Preventive programs have been offered for various cancers since themiddle of the fifties. For certain cancers even population widescreening programs exist in various developed countries. Such screeningprograms are applicable for cancer entities and the respective precursorstages such as e.g. cervical cancer, cancers of the urinary system, ofthe respiratory tract and others.

In all cases of cancer screening the outcome of a diagnostic test iscategorized in accordance with the severity of the detected lesion.Grading systems for categorizing cancers of different origins have beenestablished and are widely used. In general for any cancer type lowgrade tumors can be found that are either benign or are mostly benignwith a certain risk for progression to malignant growth.

Generally the detection of low grade lesions imposes problems to thediagnosing physician and on the patient. A moderate to high percentageof low grade lesions has the tendency to regress spontaneously withoutany further treatment necessary. On the other hand there is also a lowerpercentage of low grade lesions that will progress to more severe formsand will end up as invasive cancer. The dilemma with low grade lesionsin practice is that it is hard to predict which particular lesion isprone to regress and which will progress to invasive cancer.

Several attempts have been made to predict the risk for progression andthe potential for aggressive growth of low grade lesions. Especiallymolecular markers have been examined that might have the potential topredict progression. Especially the expression level of cell cycleregulation proteins has been checked and has been found to be helpfulunder certain circumstances.

Despite all attempts made so far there is a strong need for methods thatallow predict the progression risk for tumors and especially for lowgrade cancerous lesions and cancer precursor lesions. Therefore, it isthe object of the present invention to provide a method of predictingthe progression risk of tumors.

The inventor found that detection of overexpression of cyclin-dependentkinase inhibitors may be used to predict the progression risk of tumorsand also of early cancer precursor lesions. In addition the detection ofa combination of cyclin-dependent kinase inhibitors and of cellproliferation markers may be used for prediction of the progression riskof tumors.

SUMMARY OF THE INVENTION

It is one aspect of the present invention to provide a method forpredicting the potential for aggressive growth and the risk to progressto high grade cancer for tumors comprising determining in a cell baseddetection procedure based on the detection of the presence ofoverexpression of cyclin-dependent kinase inhibitor gene products. Incertain embodiments of the invention also the detection of specificstaining patterns of overexpression in immuno-histochemistry may beapplied in this method.

It is a second aspect of the present invention to provide a method forpredicting the potential for aggressive growth and the risk to progressto high grade cancer for tumors comprising determining in a cell baseddetection procedure performed on the basis of the detection of thesimultaneous presence of overexpression of at least one cyclin-dependentkinase inhibitor gene product and the expression of at least one cellproliferation marker gene product in at least one single cell.

It is a third aspect of the present invention to provide preparations ofprobes for diagnosis namely for predicting the progression risk and/orthe potential for aggressive growth of tumors.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 : Photomicrograph of a biopsy specimen from the cervix uteriimmuno-histochemically double-stained using monoclonal antibodiesdirected to human p16^(INK4a) protein and human Ki67 protein. Ki67rendered specific red staining, p16^(INK4a) specific brown staining. Thestaining pattern is focal for p16^(INK4a). No cells double-stained forboth markers are present.

FIG. 2 : Photomicrograph of a biopsy specimen from the cervix uteriimmuno-histochemically double-stained using monoclonal antibodiesdirected to human p16^(INK4a) protein and human Ki67 protein. Ki67rendered specific red staining, p16^(INK4a) specific brown staining. Thestaining pattern is diffuse for p16^(INK4a). Many cells double-stainedfor both markers are present.

DETAILED DESCRIPTION OF THE INVENTION

During the experiments leading to the present invention the inventorscame to the insight that detection of cyclin-dependent kinase inhibitorsin tumors and also in early cancer precursor lesions or low gradecancers may be used for prediction of the potential for aggressivegrowth of cancers. Especially overexpression of cyclin-dependent kinaseinhibitors concomitantly with expression of cell proliferation markerexpression is an indication for progression to aggressive forms ofcancer.

Further the inventors found that preparations of probes comprising a) atleast one probe specific for a cyclin-dependent kinase inhibitor geneproduct and b) at least one probe specific for a cell proliferationmarker gene product may be used for prediction of potential foraggressive growth and/or for prediction of progression risk in tumorsand in early cancer precursor lesions and low grade cancers.

The denominations given throughout this text for genes may in partrelate to the genes or proteins as they have been discovered from anyorganism. In the context of the present invention this denominationshall confer to the respective homologue of the named markers in theorganism which is particularly in question for a method as disclosedherein. In certain embodiments of the present invention this organism isa mammal and in one embodiment may be a human being. Such in oneembodiment of the present invention the named markers shall be the humanhomologues of the respective denominated ones.

A marker useful according to the present invention may be any moleculetranscribed from a gene or any molecule translated from such atranscript. Thus “gene product” as used in the context of the presentinvention may comprise polynucleotides such as e.g. DNA or RNA andpolypeptides such as proteins, proteoglycans, peptides etc.

“Cyclin-dependent kinase inhibitors” for use in the present inventioncomprise the cyclin-dependent kinase inhibitors p14^(ARF), p15^(INK4b),p16^(INK4a), p18^(INK4c), p19^(INK4d), p15^(WAF1/CIP1) and p27^(KIP1).Beside cyclin-dependent kinase inhibitors the cell cycle regulatoryprotein p14^(ARF) encoded by an alternative reading frame of thep16^(INK4a) gene may also be used for a method as disclosed herein. Forconvenience, within the context of the present invention the cell cycleregulatory protein p14^(ARF), which is by function not acyclin-dependent kinase inhibitor, shall be included in the expression“cyclin-dependent kinase inhibitor”.

“p16” or “cyclin-dependent kinase inhibitor p16^(INK4a)” as used hereinrefers to cyclin-dependent kinase inhibitor p16^(INK4a) (alsodenominated as CDKN2 or MTS1) the gene of which is located inchromosomal region 9p21. p16^(INK4a) was first described in Serrano, M.,et al., Nature, 1993 Dec. 16; 366(6456):704-7. The terms “p16^(INK4a)”or “cyclin-dependent kinase inhibitor p16^(INK4a)” in all theirgrammatical forms as used in the context of the present invention refersto nucleic acid as well as polypeptide molecules. “p16” or“cyclin-dependent kinase inhibitor p16^(INK4a)” thus comprises e.g. RNA(mRNA, hnRNA, etc.), DNA (cDNA, genomic DNA, etc.), proteins,polypeptides, proteoglycans, glycoproteins and the respective fragmentsof these molecules.

The term “(cell) proliferation marker” or “marker for cellproliferation” as used in the context of the present invention shallcomprise any marker molecule known in the art to be characteristic forthe proliferation status of cells. The proliferation status may e.g. bea status of actively proliferating cells, of retarded cellproliferation, of arrested cell proliferation, of senescent cells, ofterminally differentiated cells, of apoptosis etc. In one embodiment ofthe invention the cell proliferation marker is a marker moleculecharacteristic for active cell proliferation. In another embodiment ofthe invention the proliferation marker molecule may be a moleculecharacteristic for arrested, terminally differentiated, senescent orapoptotic cells. Generally throughout the text the term “(cell)proliferation marker” or “marker for cell proliferation” in the variousgrammatical forms is used to denominate proteins as well as nucleic acidmarkers. In case the protein name of a marker such as e.g. “replicationprotein” is used herein, this use shall be understood to bemetonymically and pertain as well to the protein as to the nucleic acidmarker molecules encoding the particular protein

In certain embodiments proliferation markers for use in the context ofthe present invention may comprise genes engaged in the DNA replicationsuch as e.g. proteins of the pre-initiation complex or of thereplication fork. Such molecules may e.g. comprise helicases, such aseukaryotic helicase or MCM proteins (MCM2, MCM3, MCM4, MCM5, MCM6,MCM7), protein TP as disclosed in WO0050451 and WO0217947 (alsodenominated HELAD1, Pomfil2, Unc-53), kinases or phosphatases engaged inthe replication process such as e.g. CDC6, CDC7, CDC7 protein kinase,Dbf4, CDC14, CDC14 protein phosphatase, CDC45 and MCM10. Furthermoreproliferation markers may comprise proteins engaged in the processivereplication fork such as e.g. topoisomerases (e.g. topoisomerase2alpha)PCNA or DNA polymerase delta, replication protein A (RPA), replicationfactor C (RFC), FEN1.

In other embodiments the proliferation markers may comprise moleculesnecessary for the maintenance of cell proliferation such as Ki67. Ki-S5or Ki-S2. In this embodiment proteins may be e.g. present throughout thewhole cell cycle. They are useful for performing a method according tothe present invention provided they are characteristic for active cellproliferation and are not significantly expressed in arrested,terminally differentiated, apoptotic or senescent states of cells. Ki67,Ki-S2 and ki-S5 as used herein shall denominate the protein markermolecules detected by the respective antibodies as well as the nucleicacids encoding these antigens.

“Tumors” as used in the context of the present invention shall refer toany kind of tumor such as e.g. benign and malignant tumors, carcinomas,sarcomas, leukemias, lymphomas, carcinomas in situ, or dysplasias.Tumors may comprise tumors of the head and the neck, tumors of therespiratory tract, tumors of the gastrointestinal tract, tumors of theurinary system, tumors of the reproductive system, tumors of theendocrine system, tumors of the central and peripheral nervous system,tumors of the skin and its appendages, tumors of the soft tissues andbones, tumors of the lymphopoietic and hematopoietic system, breastcancer, colorectal cancer, anogenital cancer etc. The term “cancer” asused in the context of the present application shall refer to cancers ofany kind and origin and precursor stages thereof, respectively.Accordingly the term “tumor” shall comprise the subject matteridentified by the terms “neoplasia”, “neoplasm”, “cancer”, “precancer”,“carcinomas in situ”, or “tumor”. Also the cytological counterpart tohistological conditions identified as “dysplastic” or as “dysplasia”shall be within the scope of the term “tumor” as used herein.

In certain embodiments of the present invention the term tumor shallrefer to early stages of tumors and shall comprise especially tumors ofGrades 1 (low grade) and 2 (intermediate grade) according to theclassification of the UICC (Union Internationale Contre le Cancer).Especially Grade 1 comprising low grade tumors are of importance for themethods of the invention. Early stages of tumors shall also refer totumors grades that correlate to the named UICC grade also in case theparticular tumor is graded according to a different grading system.Especially for Breast cancer the terms “early cancer precursor lesions”as well as “early stages of tumors” shall comprise scores 3, 4 and 5(low grade) and scores 6 and 7 (intermediate grade) of theBloom-Richardson combined scores system. For Cervical cancer the terms“early cancer precursor lesions” as well as early stages of tumors shallcomprise the categories LSIL and HSIL of the Bethesda Classificationsystem, and CIN1, CIN2 and also CIN3 of the CIN Classification system.For cytological examinations and classification according to the Munichclassification system lesions classified as Pap I, Pap II, Pap II W andPap III D shall be considered as “early cancer precursor lesions” aswell as early stages of tumors.

Tumors to which the methods of the present invention may be appliedcomprise for example, neoplastic lesions of the respiratory tract, ofthe urinary system, of the gastrointestinal tract, of the anogenitaltract, tumors associated with HPV infection and others. In oneembodiment the HPV may be a high risk HPV subtype such as HPV16, HPV18,HPV31, HPV 33, HPV35, HPV 39, HPV 45, HPV 51, HPV 52, HPV56, HPV 58, HPV59, HPV 66, and HPV 68. The tumors may be cancers of the respiratorytract, the urinary system, the reproductive tract or anogenital cancers,HPV associated cancers and particularly cervical cancer. In connectionwith the latter, its precursor stages, e.g. cervical intraepithelialneoplasias (CIN 1-3), carcinomas in situ (CIS), etc., have to bementioned particularly. The term “precursor stages” in all itsgrammatical forms as used herein comprises all precursor stages andprecursors of cancers or any other malignancies. With respect tocervical cancer precursor or preliminary stages as used herein may e.g.refer to stages of cervical intraepithelial neoplasias as identified byappropriate classification systems such as e.g. the CIN classification(CIN I-CIN III) the PAP classification (PAP I-PAP V) or the BethesdaClassification (NILM, LSIL, HSIL).

With respect to cancers of the respiratory tract cancers may compriseany malignant condition of the respiratory tract such as, e.g., cancerof the lung, the alveoles, the bronchioles, the bronchial tree and thebronchus, the nasopharyngeal space, the oral cavity, the pharynx, thenasal cavity and the paranasal sinus. Lung cancer such as small celllung cancer, non-small cell lung cancer, squamous cell lung carcinoma,small cell lung carcinoma, adenocarcinoma of the lung, large cell lungcarcinoma, adeno-squamous lung carcinoma, carcinoid tumor of the lung,broncheal gland tumor or (malignant) mesothelioma. An overview overtumors of the respiratory tract may be found in Colby T V, et al.:Tumors of the Lower Respiratory Tract, Atlas of Tumor Pathology, ThirdSeries, Fascicle 13, AFIP: Washington 1995,” which shall be incorporatedherein by reference.

Tumors of the urinary system may comprise bladder cancer, cancer of thekidney, renal pelvis, cancer of the ureters and cancer of the urethra,etc. Tumors of the reproductive system may comprise cancer andprecursory stages thereof of the ovary, the uterus, the testis, theprostate, the epididymis, etc.

The methods of the present invention may in one embodiment be applied tobreast cancer. Breast cancer may comprise any kind of carcinoma of thebreast comprising e.g. any kind of ductal carcinoma (e.g. intraductalcarcinoma in situ, invasive ductal carcinoma, medullary, mucinous(colloid), papillary, scirrhous or tubular ductal carcinoma), any kindof lobular carcinoma such as lobular carcinoma in situ, invasive lobularcarcinoma with predominant in situ component, invasive lobularcarcinoma, any kin dof nipple carcinoma (e.g. paget disease with[invasive] ductal carcinoma) and also atypical breast cancers such asphyllodes tumors and angiosarcoma.

In certain embodiments the tumors to which the method according to thepresent invention may be applied comprise any tumor characterized byoverexpression of cyclin-dependent kinase inhibitor gene products suchas e.g. p16^(INK4a); or p14^(ARF).

In certain further embodiments the methods of the invention areapplicable to HPV associated tumors. The invention in this respect isapplicable to tumors associated with HPV and especially high risk HPVtypes and mucosal HPV types. The high risk HPV may comprise HPV subtypessuch as e.g. HPV 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66 and68. Markers for HPV infection may e.g. comprise HPV expression productsof HPV genes L1, L2, E2, E4, E5, E6 or E7.

The expression “tissue or cell sample” comprises any tissue or cellsamples of any kind and nature. Examples of such tissue or cell samplesare secretions, swabs, lavages, body fluids, semen, cell- andtissue-samples, blood, smears, sputum, urine, stool, liquorcerebrospinalis, bile, gastrointestinal secretions, lymph, bone marrow,aspirates and biopsies of organs such as needle or punch biopsies and(fine)-needle aspirates. The samples may in certain embodiments comprisecervical smears, nipple aspirate fluid, bronchioalveolar lavages etc. Inparticular, smears, swabs and biopsies are indicated when the detectionof anogenital cancers, e.g. cervical cancers, is concerned. According tothe present invention cell or tissue samples may as the case may becomprise cells of the anogenital tract, of the respiratory tract or ofthe skin and its appendages. In certain embodiments the cells may becells of the uterine cervix, the vagina, the vulva, the penis, the anus,the rectum, the breast, the bronchic tree, the lung, the peritoneum, theperitoneal space, the naso-pharyngeal space, the oral cavity or theskin. In certain embodiments of the present invention the samples maycomprise cells infected by papilloma virus.

The term “biopsies” as used throughout this text shall comprise all kindof biopsies known to those of skill in the art. Thus biopsies as used inthe context of the present invention may comprise e.g. resection samplesof tumors, tissue samples prepared by endoscopic means or punch- orneedle-biopsies of organs. Biopsies comprises specimens obtained byseveral different methods such as cold knife biopsies, LEEP (loopelectrocautery excisional procedure) biopsies, etc.

Tissue or cell samples as used in the context of the present inventionmay comprise fixed or preserved cell or tissue samples. Tissue samplesmay comprise standard samples as used for histology such as e.g. frozentissue, fixed tissue, paraffin embedded tissue blocks which may be fixedby an appropriate method such as e.g. formalin fixation or any othermethod known to those of skill in the art. Cell or tissue samples maye.g. be preserved in a standard sample collection, storage ortransportation medium, known to those of skill in the art such as e.g.commercially available preservation media (formalin solution, Cytyc“PreservCyt” or “CytoLyt”, Digene “Universal Collection Medium”, TripathImaging “Cytorich”, etc.). In one embodiment of the invention the cellor tissue samples provided in standard sample collection media areliquid based cytology samples (LBC samples) which are prepared accordingto or analogous to the methods employed for cytological LBC methodsknown to those of skill in the art. Suitable cell preservation media maycontain a mixture of one or more selected from a group comprisingalcohols, aldehydes, ketones, acids, metal-ions or sublimates, ethersetc. for preservation of cellular components. Alcohols include methanol,ethanol, (n- or i-) propanol, (n-, i- or t-) butanol or higher branchedor unbranched alcohols. Aldehydes include formaldehyde, acetaldehyde,glutaraldehyde, etc. Ketones such as Acetone may be used. Acids for usein standard sample media include organic acids (acetic acid,trichloro-acetic acid, salicylic acid, picric acid) or inorganic acidssuch as e.g. chromic acid. Standard sample solutions may comprise metalssuch as silver, copper, chromium, mercury, osmium, uranium. Solutions ofsalts such as uranyl-acetate, potassiumbichromate, ammonium sulfate,etc. may be components of preservative media.

Cells preserved in suitable media (alcohols etc.) or fixed tissuesamples may be used as raw samples in the methods according to thepresent invention. In one embodiment, the tissue or cell sample may e.g.comprise a sputum sample, a cervical swab, a nipple aspirate fluid, anoral swab, an urethral swab or the like that has been transferred to apreservative medium containing alcohol.

In certain special embodiments of the present invention the sample maybe prepared as a monolayer or thin layer preparation of a cytologicalspecimen. The respective methods for preparation of monolayer orthin-layer preparation in cytology are known to those of skill in theart. In one embodiment the preparation may e.g. comprise the ThinPreptechnology. Other methods comprise conventional smears, or methodemploying suspensions of cells for preparation of the cytologicalspecimens.

Preparation of a sample may comprise e.g. obtaining a sample of atissue, of a body fluid, of cells from a patient. According to thepresent invention preparation of the sample may also comprise severalsteps of further preparations of the sample, such as preparation ofdissections, preparation of cell suspensions, spreading or applying thecells to be examined onto microscopic slides, preparation of tissuearrays, isolation of polypeptides or nucleic acids, preparation of solidphase fixed peptides or nucleic acids or preparation of beads, membranesor slides to which the molecules to be determined are coupled covalentlyor non-covalently.

In certain embodiments of the present invention the method may beperformed in an automated manner. The automation of the method may beachieved by automated staining and analysis of histological orcytological specimens on a solid surface by microscopic means. Inanother embodiment the automation may comprise a flow-cytometricanalysis of the staining of cells in solution.

The term “overexpression” in all its grammatical forms as used in thepresent invention shall refer to an expression of a particular geneproduct at a level that is elevated compared to the level of expressionin normal non-diseased cells. The term “expression” in all itsgrammatical forms as used in the present invention shall refer toexpression of a gene product at a level that is detectable and need notbe altered compared to the expression level in normal non-diseasedcells.

The method for detection of the overexpression of the cyclin-dependentkinase inhibitor gene products and of expression of the cellproliferation marker gene products according to the present invention isany method, which may (but need not) be e.g. suited to detect even verysmall amounts of specific biological molecules in biological samples.The detection reaction according to the present invention is a detectioneither on the level of nucleic acids or on the level of polypeptides.

A marker molecule is said to be detectable as used in the context of thepresent invention, provided the marker may be detected in the course ofsuitable detection procedure such as e.g. in-situ-hybridization,immuno-chemical staining, hybrid capture assay etc. The level ofexpression of a marker molecule may be made detectable using suitablereporter reactions such as e.g. a chromogenic or fluorescence basedimmuno-chemical staining or in-situ-hybridization procedure formicroscopic or automated analysis. Suitable methods for enhancing thereporter signal known to those of skill in the art may be applied in thecourse of a method according to the present invention. Thus the markeris said to be detectable in a case where the staining supersedes therespective background staining inherently obtained in theimmuno-chemical staining procedure so as to produce significant stainingresults.

The marker molecules may be detected using reagents that specificallyrecognise these molecules. The detection reaction for thecyclin-dependent kinase inhibitor gene products and/or the proliferationmarker gene products may comprise one or more reactions with detectingagents either recognising the initial marker molecules or recognisingthe prior molecules used to recognise other molecules.

In certain embodiments of the present invention two or more probes maybe used for the detection of one single marker molecule. For example twoor more different binding agents (e.g. antibodies) or oligonucleotideprobes directed against one single marker molecule (as the case may beagainst different epitopes or different sequences) may be used in thecourse of the method as disclosed herein.

The detection of the different gene products may be performed in onereaction vessel or containment or in different containmentssimultaneously or subsequently in time. Thus the different gene productsmay be detected simultaneously in one cell co-expressing both products.Otherwise cells co-expressing the gene products may be used forseparated detection reaction (separated in space or in time) to detecteach a single marker in the cells. In another embodiment there might becells expressing one or the other marker. The detection of the markermolecules in the different cells may as well be performed simultaneouslyor separately in time and/or space.

The detection reaction further may comprise a reporter reactionindicating the presence or absence and/or the level of the markermolecule gene products. The reporter reaction may be for example areaction producing a coloured compound, a bioluminescence reaction, afluorescence reaction, generally a radiation emitting reaction etc.

In certain embodiments, different marker molecules may be recognised byagents that produce different reporter signals, so that the signalsreferring to marker molecules could be distinguished. In one preferredembodiment of the invention the detection of the expression of one ofthe two or more ^(INK4a) gene products and/or proliferation marker geneproducts is carried out simultaneously. In this case the reporterreaction may for example employ different fluorescent or chromogeniclabels for the different molecules detected.

However within the context of the present invention it must notnecessarily be answered whether the one or the other proliferationmarker or cyclin-dependent kinase inhibitor gene product is expressed inthe cells. In certain embodiments the question is whether anyproliferation marker and/or cyclin-dependent kinase inhibitor geneproduct is expressed. Such in the course of the experiments a proceduremay be chosen, that gives the same fluorescence or chromogenic signal asindication of the presence of a proliferation marker. This procedure issuitable to improve sensitivity of the detection of the cellproliferation characteristics (different markers characteristic foractive cell proliferation). As the case may be the procedure may beapplied as to render one detectable signal for two, three, four or evenmore marker molecules being characteristic for cell proliferation.Analogous the same may under certain circumstances be true for thecyclin-dependent kinase inhibitor gene products. It must be understood,that as the case may be different staining signals for differentproliferation marker molecules may be desirable. The procedures may beapplied to the necessities of the respective experiment.

In certain embodiments of the present invention a combination of one ormore (e.g. two different) cyclin-dependent kinase inhibitor geneproducts may be detected with a combination of one or more e.g. a set oftwo, a set of three, a set of four, a set of five or a set of even moremarkers for cells proliferation. As the case may be the detection of themarker molecules for cell proliferation may render only one reportersignal. In other cases each single marker for cell proliferation mayrender a specific reporter signal or groups of marker molecules mayrender specific reporter signals.

Signals for the indication of the presence of immuno-reactivity may bechromogenic signals produced by various methods known in the art. Suchmethods may for example comprise the generation of coloured precipitatesat the site of antigen using horseradish peroxidase for enzymaticconversion and DAB, AEC, HISTOMARK® ORANGE, HISTOMARK® BLACK, etc. as achromogen, or using the alkaline phosphatase enzyme for conversion of achromogen and e.g. BCIP-NBT, Fast Red, New Fuchsin, etc. as chromogen.Alternatively or even in combination fluorescent signals may be used.Suitable reporter signals comprise fluorescent labels such asfluorescein, rhodamin etc.

Applicable formats for the detection reaction according to the presentinvention may be, blotting techniques, such as Western-Blot,Southern-blot, Northern-blot, immuno-cytochemical orimmuno-histochemical procedures. The blotting techniques are known tothose of ordinary skill in the art and may be performed for example aselectro-blots, semidry-blots, vacuum-blots or dot-blots.Immuno-cyto/histochemical staining procedures are known to those ofskill in the art and may comprise binding agent mediated detection ofpolypeptides as well as in situ hybridisation techniques. Both differenttechniques may even be applied simultaneously. In certain embodimenthybrid capture of nucleic acids may be used for the detection.Amplification reaction may also be applicable for the detection of e.g.nucleic acid molecules.

In one embodiment of the invention the detection of the level ofcyclin-dependent kinase inhibitor and/or proliferation marker geneproducts is carried out by detection of the respective nucleic acids(e.g. mRNA) or fragments thereof present in the sample. The means fordetection of nucleic acid molecules are known to those skilled in theart. The procedure for the detection of nucleic acids can for example becarried out by a binding reaction of the molecule to be detected tocomplementary nucleic acid probes, proteins with binding specificity forthe nucleic acids or any other entities specifically recognising andbinding to said nucleic acids. In one embodiment in situ hybridisationof oligonucleotide probes to nucleic acids in a sample may be used forthe detection of expression products or markers.

Probes as used in the context of the present invention may be any agentbinding specifically to a molecule and shall include nucleic acidprobes, peptide and protein probes and other probes. In the case ofnucleic acids a probe may be an oligonucleotide hybridising to aparticular sequence. In one embodiment the probe may be e.g. a primer.In the case of the detection of polypeptides or proteins the probe asused herein may be e.g. a binding agent such as an antibody. In certainembodiments of the present invention the probes may be detectablylabelled. The label may be selected from the group comprising aradioisotope, a bioluminescent compound, a chemiluminescent compound, afluorescent compound, a metal chelate, or an enzyme. Probes may beapplied in any detection procedure known in the art e.g. in the courseof an in situ hybridisation procedure, in the course of hybrid captureassays, in the course of immuno-chemical staining reaction, in thecourse of blotting techniques etc.

This method may be performed as well in vitro as directly in situ forexample in the course of a detecting staining reaction. Another way ofdetecting the marker mRNAs in a sample performed in the method accordingto the present invention is an amplification reaction of nucleic acids,which can be carried out in a quantitative manner such as for examplethe polymerase chain reaction. In a preferred embodiment of the presentinvention real time RT PCR may be used to quantify the level of markermRNAs in samples of dysplasias or tumors (cells or tissue samples).

In another preferred embodiment of the invention the detection of thelevel of cyclin-dependent kinase inhibitor and/or proliferation markergene products is carried out by determining the level of expression of aprotein or fragments thereof. The determination of the marker geneproduct on the protein level can for example be carried out in areaction comprising a binding agent specific for the detection of theparticular marker polypeptide.

The binding agents can be used in many different detection techniquesfor example in western-blot, ELISA or immuno-precipitation. Generallypolypeptide binding agent based detection can be carried out as well invitro as directly in situ for example in the course of animmuno-chemical staining reaction. Immunochemical staining reaction may,as the case may be, be applied in histological and cytological stainingprocedures. Any other method for determining the amount of particularpolypeptides in biological samples can be used according to the presentinvention.

The immuno-cytochemical (comprising inter alia histology and cytologyapplications) staining and/or imaging procedures for use in the contextof the present invention may comprise e.g. the staining of cytologicalor histological preparations with chromogenic or fluorescent dyes. Thestaining may e.g. comprise binding of the molecules to be detected by afirst binding agent, which itself is detected by a secondary bindingagent, which may be labelled. The first binding agent may in certainembodiments be a nucleic acid or a protein binding agent (e.g. anantibody) and the secondary binding agent may be e.g. a secondaryantibody recognizing the first binding agent.

Any methods known in the art for performing staining of cytochemical orhistochemical staining may be applied in the course of a methodaccording to the present invention.

According to the present invention binding agents may be used isolatedor in combination. By means of combination it is possible to achieve ahigher degree of sensitivity. The term antibody, preferably, relates toantibodies which consist essentially of pooled monoclonal antibodieswith different epitopic specificities, as well as distinct monoclonalantibody preparations. Polyclonal antibody preparations may be usedalternatively according to the present invention.

Binding agents as used in the context of the present invention for thedetection of the level of cyclin-dependent kinase inhibitor polypeptidessuch as p16^(INK4a) or p14^(ARF) polypeptides and proliferation markerpolypeptides such as e.g. MCM5, MCM2, Ki67, Ki-S5, PCNA or Ki-S2polypeptides may comprise antibodies. An antibody or antigen-bindingagent is said to react specifically, if it reacts at a detectable levelwith a protein disclosed herein, and does not significantly react withother proteins. The term “antibody” in all its grammatical forms as usedin the context of the present invention shall comprise any kind ofantibody including monoclonal and polyclonal antibodies, antigen-bindingfragments, antibody fragments, fab′ fragments, bi-functional hybridantibodies, single chain antibodies, humanized antibodiespeptidomimetics containing minimal antigen-binding epitopes,anti-cullines (Anti-Caline™) etc.

Monoclonal antibodies are raised against antigen containing fragments ofthe polypeptide of the invention using any of a variety of techniquesknown to those of ordinary skill in the art; see, e.g., Harlow and Lane,Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, 1988. Inone such technique, an immunogen comprising the antigenic polypeptide ora synthetic part thereof is initially injected into any of a widevariety of mammals (e.g., mice, rats, rabbits, sheep and goats). In thisstep, the polypeptides of this invention may serve as the immunogenwithout modification. Alternatively, particularly for relatively shortpolypeptides, a superior immune response may be elicited if thepolypeptide is joined to a carrier protein, such as bovine serum albuminor keyhole limpet hemocyanin. The immunogen is injected into the animalhost, preferably according to a predetermined schedule incorporating oneor more booster immunizations, and the animals are bled periodically.Polyclonal antibodies specific for the polypeptide may then be purifiedfrom such antisera by, for example, affinity chromatography using thepolypeptide coupled to a suitable solid support.

The cyclin-dependent kinase inhibitor gene products and/or proliferationmarker gene products may according to the present invention be detectedsimultaneously. In this context simultaneously according to the presentinvention shall mean either literally at the same instant or within thesame testing procedure, whereby the single detection steps aretemporarily consecutive.

The detection procedure according to the present invention mayfurthermore comprise a cytochemical staining procedure rendering achromogenic or fluorescent staining of cells or cell compartments. Suchstaining procedures are known to those of skill in the art and may forexample comprise e.g. staining for acidophilic or basophilic structures,of subcellular regions (e.g. the nucleus, the mitochondria, the golgi,the cytoplasm etc.), of specific molecules (of chromosomes, of lipids,of glycoproteins, of polysaccharids etc.) in the cytological specimens.Fluorescence dyes such as DAPI, Quinacrin, Chromomycin, etc. may beemployed. Furthermore chromogenic dyes such as Azan, Acridin-orange,Hematoxylin, Eosin, Sudan-red, Thiazin-stains (Toluidin-blue, Thionin)may be applied. In other embodiments staining procedures such asPap-staining, Giemsa-staining, Hematoxylin-Eosin staining, van-Giesonstaining, Schiff-staining (using Schiff reagent), staining proceduresemploying precipitation of metals (such as e.g. of silver in stainingprocedures employing Silver Nitrate) or insoluble stains such as e.g. ofTurnbulls-blue (or other insoluble metal cyanides), etc. may be used inthe course of a method as disclosed herein. It must be understood, thatthe named dyes and staining methods shall be examples for the applicablemethods and that any other method known in the art may be applied to amethod as disclosed herein.

The staining procedures may produce chromogenic stains for lightmicroscopic inspection or fluorescent stains for inspection underfluorescence microscopic conditions. In another embodiment of thepresent invention radiation emitting procedures, procedures employingsubstances impairing the transmission of radiation or other contrastmedia for imaging of the cytological conditions in a sample (e.g. thegeneration of optical impression by means such as(micro-)autoradiographic or (micro-)radiographic picture generation) maybe of use for a method according to the present invention.

All the staining and imaging procedures may be used for analysis notonly in microscopic procedures but also in automated analysis proceduressuch flow cytometry, automated microscopic (computerized or computeraided) analysis or any other method for analysis of stained cytologicalspecimens.

The analysis of the staining or imaging results of the differentprocedures may be performed in a single analysis step or in differentsubsequent steps. E.g. the light microscopic inspection of a specimenmay be performed before or after fluorescence microsopic inspection ofthe specimen. In Fluorescence microscopy the analysis of differentstains with different excitation wavelengths may be analysessimultaneous or subsequently. Other imaging methods may be employedsimultaneously or subsequently to the named procedures.

There may be various circumstances, under which combinations ofdifferent staining methods will be suitable. E.g. in cases, where nosatisfying cytological staining results may be achieved byimmuno-chemical staining the additional application of generalcytological staining techniques may be suitable.

Analysis of staining results according to the present invention incertain embodiments of the invention comprises the detection of certainstaining patterns in histological procedures. Generally stainingpatterns can be classified in various ways. Any suitable classificationof staining patterns in histology shall be applicable for the purpose ofthe present invention. Especially the detection of diffuse stainingpattern shall be of relevance for the invention. A “diffuse stainingpattern” refers to a histological staining of an epithelium whereimmunochemical staining for a particular gene product is detected as acontinuous staining of cells of the basal and parabasal cell layers ofthe squamous cervical epithelium, with or without staining of cells ofsuperficial cell layers. In certain embodiments the detection of diffusestaining pattern for cyclin-dependent kinase inhibitors may be the basisfor prediction of progression risk or of potential for aggressive growthof tumors. Diffuse staining pattern is to be discriminated from focalstaining patterns, where only staining of isolated cells or small cellclusters; i.e., a non-continuous staining, particularly not of the basaland parabasal cells is observed.

Preparations as used in the context of the present invention shall referto a chemical preparation comprising more than one substance. Inparticular the preparations of the present invention are preparationscomprising at least two different probes one of them being specific fora cyclin-dependent kinase inhibitor gene product at least one other ofthem being specific for a cell proliferation marker gene product. Inaddition the preparation according to the present invention may comprisefurther substances such as preservatives, stabilizers, buffers,diluents, and others. The substances used and the compositions suitablefor providing probes are known to those of skill in the art. In certainembodiments of the present invention the probes may be nucleic acidprobes or antibodies. Polyclonal and/or monoclonal antibodies forincorporation into the preparation of the invention may be derived fromany suitable animal known to those of skill in the art including withoutlimitation mouse, rat, hamster, goat, rabbit, human, horse, cow, pig,etc. Also synthetic or genetically engineered antibodies or antibodiesgenerated in transgenic plants, microorganisms or animal may beincorporated. In certain embodiments of the invention the probes may bemonoclonal antibodies. In further embodiments the antibodies may bechosen in a way that the antibodies specific for cyclin-dependent kinaseinhibitors are derived from one animal (e.g. mouse, rat or the like) andthe antibodies specific for cell proliferation marker are chosen fromanother animal (e.g. goat, rabbit etc.)

In one embodiment the preparation may for example be a preparation ofp16^(INK4a) specific antibodies together with Ki67 specific antibodies.

The present invention provides methods for predicting the potential foraggressive growth and the risk to progress to high grade cancer fortumors comprising determining in a cell based detection procedure basedon the detection of the presence of overexpression of cyclin-dependentkinase inhibitor gene products. In certain embodiments of the inventionalso the detection of specific staining patterns of overexpression inimmuno-histochemistry may be applied in this method. Further the presentinvention provides methods for predicting the potential for aggressivegrowth and the risk to progress to high grade cancer for tumorscomprising determining in a cell based detection procedure performed onthe basis of the detection of the simultaneous presence ofoverexpression of at least one cyclin-dependent kinase inhibitor geneproduct and the expression of at least one cell proliferation markergene product in at least one single cell. Even further the presentinvention provides preparations of probes for diagnosis namely forpredicting the progression risk and/or the potential for aggressivegrowth of tumors. The invention solves the problem present in the artthat well suited tools for prognosis of growth characteristics and ofthe progression risk of tumors were not sufficiently available. Thepresent invention now puts the skilled person in the position to predictprognosis and the potential for aggressive growth. The methods andpreparations of the present invention accordingly may form the basis forimprovement in patient care and in tailoring adequate therapy forpatients.

EXAMPLES

The following examples are given for the purpose of illustration onlyand are not intended to limit the scope of the invention disclosedherein.

Example 1

Prediction of the Progression Risk of Histological Specimens Obtainedfrom the Cervix Uteri Classified as CIN 1 by Detection of p16^(INK4a)Protein and Ki67 Protein

Archival histological samples (sections generated from neutral bufferedformalin fixed, paraffin embedded punch biopsies) of the cervix uteriwere immuno-histochemically stained using antibodies specific forp16^(INK4a) and Ki-67. In total 10 specimens have been stained.

Specimens with available follow up data have been chosen. For 6specimens follow up examinations revealed regression of the CIN 1. For 4specimens follow up examinations revealed progression of the CIN1lesions to high grade dysplastic lesions. For all 10 specimens also HPVdata were available or were generated during the experiments performed.

All specimens were treated as follows.

Tissue blocks were sectioned into slices of 4 m thickness. Prior todeparaffinization slides have been placed in a drying oven at atemperature of 60° C. for a time period of 30 minutes to melt theparaffin. Tissue slides have then been de-paraffinized to removeembedding medium have been rehydrated before the staining procedure wasbe performed. Subsequently Epitope retrieval was performed for 10minutes at 98° C. using 10 mM Tris as Epitope Retrieval Solution.Staining of the slides has been performed with a mixture of primaryantibodies, i.e. monoclonal mouse-anti-human-p16^(INK4a) and monoclonalrabbit-anti-human-Ki67. For detection secondary reagents based on apolymeric support coupled to the respective secondary antibodies and toalkaline phoshpatase (for the anti-rabbit secondary antibody) and tohorseradish peroxidase (for the anti-mouse secondary antibody) have beenused. The staining was performed on a Labvision Autostainer Instrumentusing the following program:

200 μL Peroxidase-Blocking Reagent—5 minutes;

200 μL Primary Antibody Solution—30 minutes;

200 μL Visualization Reagent HRP—15 minutes;

200 μL Visualization Reagent AP—15 minutes;

200 μL Substrate-Chromogen Solution (DAB)—10 minutes;

200 μL Substrate-Fast Red (Fast Red)—15 minutes;

Between and after the single steps appropriate rinse steps wereperformed. After staining counterstain with hematoxylin was performedand slides were permanently mounted.Photomicrographs of the slides are given in FIGS. 1 to 2 .Microscopic inspection of the slides revealed that those CIN 1 lesionsthat had proven to regress spontaneously showed no cells double-stainedfor p16^(INK4a) and Ki67 (cf. FIG. 1 ). Further these lesions showedsporadic or focal staining pattern for p16^(INK4a) and no diffusestaining pattern (cf. also FIG. 1 ).

A correlation between high risk HPV results and p16 staining inimmuno-histochemistry and p16^(INK4a)/Ki67 double staining inimmuno-histochemistry for CIN that showed spontaneous regression isgiven in the following tables:

TABLE 1 Correlation of p16^(INK4a) staining results with hr HPV statusfor CIN1 lesions that had proven to spontaneously regress. p16 (+) p16(−) Σ hr HPV+ 1 4 5 hr HPV− — 1 1 Σ 1 5 6

TABLE 2 Correlation of p16/Ki67 double-staining results with hr HPVstatus for CIN1 lesions that had proven to spontaneously regress.p16/Ki67 (+) p16/Ki67 (−) Σ hr HPV+ — 5 5 hr HPV− — 1 1 Σ — 6 6

p16^(INK4a) is considered positive (+) for the purpose of the abovetables once there is a positive p16^(INK4a) immuno-reactivityirrespective of the staining pattern observed. p16^(INK4a) and Ki67double staining is considered positive (+) in case there is at least onesingle cell observed in the specimen that is positive for both markersand accordingly is double-stained.

In contrast all of the four lesions that had proven to progress to highgrade lesions show double-staining for p16^(INK4a) and Ki67 in at leastone single cell in the specimen. Furthermore such specimens showedstrong diffuse staining pattern for p16^(INK4a) alone (cf. FIG. 2 ).

A correlation between high risk HPV results and p16^(INK4a) staining inimmuno-histochemistry and p16^(INK4a)/Ki67 double staining inimmuno-histochemistry CIN1 with progression potential is given in thefollowing tables:

TABLE 3 Correlation of p16^(INK4a) staining results with hr HPV statusfor CIN1 lesions that had proven to progress to high grade lesions. p16(+) p16 (−) Σ hr HPV+ 4 — 4 hr HPV− — — — Σ 4 — 4

TABLE 4 Correlation of p16/Ki67 double-staining results with hr HPVstatus for CIN1 lesions that had proven to progress to high gradelesions. p16/Ki67 (+) _(P)16/Ki67 (−) Σ hr HPV+ 4 — 4 hr HPV− — — — Σ 4— 4

Again p16^(INK4a) is considered positive (+) for the purpose of theabove tables once there is a positive p16^(INK4a) immuno-reactivityirrespective of the staining pattern observed. p16^(INK4a) and Ki67double staining is considered positive (+) in case there is at least onesingle cell observed in the specimen that is positive for both markersand accordingly is double-stained.

It could be concluded, that as on the basis of the p16^(INK4a) stainingpattern prediction of the progression risk of CIN 1 lesions would havebeen facilitated. However it must be noted, that the interpretation ofthe staining pattern of histological preparation leaves room forsubjective interpretation and is therefore to somewhat extent prone toerrors. As an alternative that is less prone to subjectiveinterpretation the double staining method is provided. Also in this caseon the basis of the presence of cells double-stained for p16^(INK4a) andKi67 it would have been possible to predict those CIN 1 lesions testedthat had the potential to progress to high grade lesions. For hr HPVtest the results show that 9 out of 10 tested specimens were hr HPVpositive. So 5 out of 6 of the specimens which spontaneously regressedshowed hr HPV positivity.

The experiment shows that determination of the progression risk of CIN1lesions was possible on the basis either of p16^(INK4a) staining pattern(diffuse staining pattern is indicative of a risk to progress to highgrade lesions), on the basis of double staining of cells for p16^(INK4a)and Ki67 or on a combination of hr HPV with p16^(INK4a) positivity. Forthe double staining the prediction of the risk for progression is alsogiven when combining the double staining test with hr HPV testing.

Example 2

Prediction of the Progression Risk in Breast Biopsies by Detection ofp16^(INK4a) Protein and Different Cell Proliferation Marker Proteins

In total 15 archival biopsy specimens of breast tumors (ductal carcinomain-situ and lobular carcinoma in situ specimens) were used for thepresent experiment. 10 of the tested specimens were of less aggressivetumors, 5 stem from more aggressive tumor types. Progression risk andaggressiveness of the tumors were proven by follow up data on theindividual cases. For each sample five sections were subjected todifferent double staining experiments. In each experiment p16^(INK4a)staining was combined with one of the following cell proliferationmarkers: Ki67, MCM2, KiS2, MCM5, topoisomerase-2-alpha.

Specimen preparation and staining was performed as described above forExample 1.

Also in this experiment microscopic inspection revealed, thatdouble-staining of single cells was detected only in those specimensthat had more aggressive characteristics and had unfavourable prognosis.The five specimens characterized by the more aggressive growth as provenby follow up data showed diffuse staining pattern for p16^(INK4a) and inaddition also are characterized by many double stained cells showingKi67 staining together with p16^(INK4a) staining in single cells. Forthe other double staining experiments each of the tested cellproliferation markers showed in at least 4 out of the 5 specimens atleast one single cell with double staining. A combination of any of thetested cell proliferation markers with p16^(INK4a) accordingly is suitedto detect those tumors of the breast that have the potential foraggressive growth that need more rigorous therapy. To enhance thesensitivity for double staining a combination of more than oneproliferation marker could be considered to be included in combinationwith the cyclin dependent kinase inhibitor.

For the specimens where follow up had proven less aggressive growthproperties and more favourable prognosis p16 overexpression could beshown as well. However the staining pattern was not diffuse here.Further staining for p16^(INK4a) and for the proliferation markers couldbe detected in the overall specimen however not as a double staining ofa single cell.

Accordingly as well the staining pattern for the cyclin dependent kinaseinhibitor as well as the double staining of single cells with bothmarkers is indicative for the more aggressive growth properties.

What is claimed is:
 1. A method for predicting a potential for breasttumors for aggressive growth and/or a risk to progress to high gradecancer, comprising: obtaining a breast tumor sample of a subject;measuring by a cell based detection procedure whether at least onesingle cell of the breast tumor sample simultaneously overexpressesp16^(INK4a) and expresses Ki67; and determining that the tumors have apotential for aggressive growth and/or a risk to progress to high gradecancer when there is at least one single cell in the sample having thesimultaneous presence of overexpression of p16^(INK4a) and theexpression of Ki67.
 2. The method of claim 1, wherein the cell baseddetection procedure is immunohistochemistry or immunocytochemistry. 3.The method of claim 1, said measuring uses antibodies againstp16^(INK4a) and Ki67.
 4. The method of claim 1, wherein the sample is atissue sample or a cell sample.
 5. The method of claim 1, wherein saidbreast tumors are early cancer precursor lesions or low grade cancers.6. The method of claim 1, wherein said breast tumors are carcinoma insitu, dysplasia, or neoplasia.